Soft surfaces are a feature of many natural phenomena, particularly when immersed in liquid, including cell membranes and immiscible liquid droplets. Many imaging and measurement techniques used for the study of such surfaces employ a probing method that applies forces which may induce errors by disturbing the surface under observation or which require modification of the surface before such observation can be carried out.
The cell is the most fundamental unit of living organisms, whether animal or plant. The study of its structure and composition, and how its various constituents function, lends valuable insight into the complex processes that occur in integrated biological systems. This requires techniques that allow investigation of cell samples to be conducted in real-time, non-invasively, and in solutions that mimic physiological conditions so that cell functionality is retained.
Optical microscopy (using visible light) has been applied widely to study live cells. However, the resolution is limited by diffraction to about 200-250 nm. For more detailed study, one commonly used method is electron microscopy, where it is possible to obtain images with 10 nm resolution, but the sample needs to be fixed prior to imaging. Hence, it is not possible to use an electron microscope to study living cells.
Another possible high resolution technique is based on the use of scanning probe microscopy (SPM), in which a sharp probe tip is scanned in close proximity to the sample under study. The consequent interactions and thus the chemical/physical properties of the sample can be plotted as a function of the tip's position with respect to the sample, to generate a profile of this measured interaction. Members of the SPM family that are commonly applied to biological imaging are atomic force microscopy (AFM), scanning ion-conductance microscopy (SICM) and scanning near-field optical microscopy (SNOM).
Atomic force microscopy (AFM) is commonly used to study the response of a surface to mechanical force or pressure. When using AFM, the tip cantilever spring constant affects how much the surface under study will be displaced by the measurement or detection process and sets a limit to the softness of a surface which can be studied. An additional difficulty with AFM when used in contact or tapping mode is the likelihood of the surface adhering to the probe tip, altering the measurements during retraction and leading to contamination of the tip and mechanical damage to the surface.
In other cases the environment required by the probing method requires modification of the surface before imaging, as with electron microscopy where the need for a vacuum or low pressure gas may require stabilization of the surface and removal of liquid before imaging may be carried out.
Scanning ion conductance microscopy (SICM) is a form of scanning probe microscopy (SPM) that allows the high resolution imaging of soft surfaces without any contact or force interaction whatsoever and in the normal liquid environment of the subject. In SICM, typically an electrolyte-filled, glass micropipette is scanned over the surface of a sample bathed in an electrolytic solution; see Hansma et al (1989) Science 243:641-3. A quartz pipette may also be used. WO-A-00/63736 discloses that SICM can be used effectively, e.g. to scan the surface of a live cell by controlling the position of such a probe. This is achieved by adjusting the distance of the tip of the micropipette from the surface so as to maintain the current at a constant value, typically that which keeps the probe at a distance of some nanometers from it. The pipette-sample separation is maintained at a constant value by monitoring the ion-current that flows via the pipette aperture. The flow is between two electrodes: one inside the pipette and another outside in the electrolyte solution. For an applied bias between the electrodes, the ion-current signal depends on a combination of the micropipette's resistance (RP) and the access resistance (RAC) which is the resistance along the convergent paths from the bath to the micropipette opening. RP depends on the tip diameter and cone angle of the micropipette, whereas RAC displays complicated dependence on the sample's electrochemical properties of the bath and the sample, geometry and separation from the probe. It is RAC that lends ion-current sensitivity to the pipette-sample separation and allows its exploitation in maintaining the distance such that contact does not occur.
The optimum tip-sample separation that has allowed SICM to be established as a non-contact profiling method for elaborated surfaces, is approximately one-half of the tip diameter; see Korchev et al (1997), J. Microsc. 188:17-23, and also Biophys. J. 73:653-8. The outputs of the system controlling the position of the tip are used to generate images of topographic features on the sample surface. The spatial resolution achievable using SICM is dependent on the size of the tip aperture, and is typically between 50 nm and 1.5 μm. This produces a corresponding resolution.
The sensitivity of the micropipette used in SICM is highest to surfaces directly below the micropipette tip and is less so to surface structures that lie at the sides of the tip. If the micropipette is scanned across a surface whose features are of a similar scale to the diameter of the micropipette tip, the SICM system is able to keep the tip out of contact with the surface. However, if the surface contains features and structures of a height much larger than the tip diameter, and which include steep edges or walls, then the scanning speed on the SICM system must be reduced in order to avoid collision, resulting in a longer time spent at each point on the surface. In the extreme, where the target surface is a convoluted structure such as a cluster of interwoven neurons, or a matrix or scaffold within which cells are growing, there is a risk that the micropipette would become entangled and that the SICM system would be unable to scan.
WO-A-00/63736 discloses a method by which the probe is scanned across the surface at a fixed speed or rate. Other methods are known in which the time spent by the probe at any point is varied to allow the probe ion current and vertical position to stabilise within a given range. In all of these methods, however, the resolution of the scan or density of points measured during the scan, as represented by the number of points measured per scan line and the number of lines scanned per image, is constant throughout that scan. As a result, an increase in the time spent at each point leads to a proportionate increase in the total scan time. The time to generate a high resolution image of a convoluted surface may become so long as to be prohibitive.
Previous methods to confine the scan to the most relevant region have been based on optical microscopic surveys, including computer analysis of the resulting images (NASA/TM-2004-213383). This approach can be complementary, but is limited to a resolution governed by the wavelength of the light.
Mann et al, J. Neuro. Methods, 2003; 116: 113-117, discloses the technique of pulse-mode scanning ion-conductance microscopy. This is used to control the distance between the SCIM probe and the surface. The technique uses constant current pulses to monitor the change in resistance.
Happel et al, J. Microscopy, 2003; 212: 144-151, discloses the use of a pulse-mode scanning ion conductance microscopy to observe volume changes and cell membrane movements during the locomotion of cultured cells. The microscope apparatus uses current pulses to control the difference between the cell surface and the electrode tip as well as a back-step to prevent contact of the tip with the cell membrane during lateral movements of the probe. The apparatus is used with a constant resolution to determine areas having high surface structures. Lateral scans can then be performed at different heights depending on the expected height of the surface structures. Although this method has advantages, it still results in low scan speeds.